Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/20—Oxygen containing
  • Y10T436/206664—Ozone or peroxide

Definitions

• the present invention is concerned with process and diagnostic agent for the determination of hydroperoxides and of substances from which hydrogen peroxide or other hydroperoxides are liberated by a previous reaction, as well as for the determination of peroxidase and of other peroxidatively active substances.
• glucose in urine, blood and serum is, in the case of, diabetes, of especial diagnostic interest, as is also the detection of peroxidatively active substances, such as hemoglobin, in urine and blood, and the detection of hydroperoxides in, for example, the milk industry, the cosmetics industry and in polymer chemistry.
• a series of compounds which are oxidized to dyestuffs with hydrogen peroxide and peroxidase as catalyst.
• Preferred compounds of this type include, for example, the Nadi reagents, p-phenylene-diamine derivatives, carboazines, guaiac resins, aldazine and the like; Previously, it has been preferred to use benzidine, o-dianisidine and o-tolidine. However, some of these compounds are not very stable and, according to recent findings, can also be a health hazard so that their use does not appear to be free of danger.
• R is lower alkyl
• R is hydrogen or alkyl, preferably lower alkyl, or, together with Y, represents a fused benzene or napthalene nucleus
• X is sulfur or oxygen or an alkylated imino group, preferably carrying lower alkyl groups, or a vinylene radical
• Y is a methine radical which, together with R,, can also form a benzene or naphthalene nucleus, are absolutely stable and are particularly suitable for the determination of hydrogen peroxide and of other hydroperoxides, notonly by means of an optical test but also with the help of reagent papers and reagent films.
• Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V is hydrogen or amino or hydroxyl; ⁇ V is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as with the alkali metal salts thereof.
• a process for the determination of hydroperoxides and of substances which react with the liberation of hydrogen peroxide, as well as of peroxidase and of peroxidatively active substances, with the help of a chromogen using the reaction of the hydroperoxides with peroxidase or peroxidatively active substances, by evaluation, in known manner, and of the coloration, wherein a compound of general formula I is used as chromogen, preferably in admixture with a compound of general formula ll.
• the evaluation of the coloration can be carried out, for example, by optical measurement in a spectrophotometer or, in the case of the use of test paper strips and test films, by comparison of the color strength with blank samples of known composition or with color charts.
• hydroperoxides by the process according to the present invention is particularly useful for coupled and uncoupled enzyme reactions, for example, for the determination of glucose, galactose, amino acids, uric acid, peroxides, hemoglobin, peroxidase or other peroxidatively active substances, as well as of enzyme activities. Because of their outstanding importance, the routine determination of substrates of this type is now an essential feature of clinical chemistry and of foodstuff chemistry.
• glucose-oxidase In the case of the determination of glucose, the latter is, for example, oxidized by glucose-oxidase to gluconic acid, atmospheric oxygen thereby being reduced to hydrogen peroxide.
• the hydrogen peroxide By means of peroxidase or of a peroxidatively active substance, the hydrogen peroxide then oxidizes the indicator used according to the present invention to give the corresponding color dyestuff.
• analytically useful enzyme systems of this type which react with the liberation of hydrogen peroxide, include L-amino acid oxidase L-amino acids, uricase uric acid, xanthine oxidase hypoxanthine or xanthine, glycine oxidase glycine, monoamine oxidase monoamine (such as adrenaline, mescaline and the like), diamine oxidase diamine (such as histamine), luciferase luciferin, D-aspartic acid oxidase D-aspartic acid, liver aldehyde oxidase aldehyde, galactose oxidase galactose, Edsons flavine enzyme +lactic acid.
• a diagnostic agent for the determination of hydroperoxides and of substances which react with the liberation of hydrogen peroxide which comprises peroxidase or a peroxidatively active substance and a chromogen of general formula I alone or in admixture with a compound of general formula ll.
• a diagnostic agent for the determination of hemoglobin and of other peroxidatively active substances which comprises hydrogen peroxide or a substance forming hydrogen peroxide and a chromogen of general formula I alone or in admixture with a compound of general formula ll.
• a substance forming hydrogen peroxide is intended to mean not only a single compound, such as an organic peroxide, but also a mixture, such as glucose and glucose-oxidase.
• the new diagnostic agents according to the present invention can be prepared in the form of solutions in appropriate solvents, if necessary with the addition of conventional adjuvants, such as buffers.
• the new diagnostic agents can be prepared in the form of test papers by the impregnation of suitable absorbent materials, such as filter paper, with solutions of the components of the diagnostic reagents or can be used for making reagent films.
• suitable absorbent materials such as filter paper
• the compounds of general formula I can be prepared in known manner, for example, by the reaction of 2-haloor 2- merapto-quatemary salts with an appropriate N-alkylated hydrazone salt, with the addition of an amine, preferably of triethylamine, in a polar solvent, for example methanol, at a temperature of l 5l00 C. Depending upon the reactivity of the individual reaction components, the reaction is finished after a period of from minutes to 2 hours (see S. Hi'Inig et al. Liebigs Ann.
• EXAMPLE 1 Reagent Film for the Detection of Glucose in Blood Forty-Five g. Propiofan (BASF), 35 g. of a solution of 37 g. algipon in 2 liters of a 0.5 M phosphate or citrate buffer of pH 5.7, l g. Texapon P (Henkel, Dusseldorf), 10 ml. water, 75
• EXAMPLE 2 To a mixture of Propiofan, algipon, buffer, Texapon P, water, peroxidase and glucose oxidase prepared in the manner described in example I, there were added 8 ml. of a concentrated methanolic solution of l-[ l-methyl-benzo(f)quinoline- 2-one]-2-[ 1ethyl-quinoline-2-onel-azine, as well as 4 ml. of a 2 percent solution of p-aminosalicylic acid. A foil was coated with the mixture prepared in this manner with a layer thickness of 300 u and then of When glucose-containing blood was dropped onto the reagent film thus produced, left to react therewith and the blood then again removed, there was obtained a pink color reaction.
• EXAMPLE 4 A filter paper (Schleicher & Schilll 23 S) was impregnated with a solution of 0.25 ml. Texapon P (Henkel, Dusseldorf) and mg. sodium alginate in 100 ml. of a 0.1M phosphate buffer of pH 5.7 and then dried. The buffered paper thus prepared was further impregnated with a 0.01 percent methanolic solution of l-[l-methyl-benzo-(f)quinoline-2- one]-2 -[1-ethyl-quinoline-2-one]-azine and a 0.01 percent methanolic solution of p-aminosalicylic acid and again dried. When a drop of a body fluid which contained blood was applied to a test paper strip prepared in this manner, together with a drop of a 3 percent hydrogen peroxide solution, then the paper became blue-violet colored.
• EXAMPLE 5 Dection of Glucose in Urine .Eil QKBEI P .l9hlll3[.&?hiil wa pr n with a solution of 75 mg. peroxidase, 143 mg. glucose oxidase and 0.25 ml. Texapon P in 100 ml. ofa 0.1M phosphate buffer of pH 5.6 and dried.
• the enzyme-buffer paper thus prepared was subsequently impregnated with a 0.01 percent solution of 1-[ l-methyl-benzo(f)quinoline-2-one]-2-[ 3-ethyl-benzoxazol-Lonel-azine in chloroform and with a 0.0] percent methanolic solution of p-aminosalicylic acid and again dried.
• a reagent paper prepared in this manner was dipped into a urine with a pathological glucose concentration, then the reagent paper assumed a red-brown coloration.
• EXAMPLE 6 Detection of Glucose with and without a Modifier Substance of General Formula ll
• 10 ml. methanol which, if desired, contained 0.1 percent p-aminosalicylic acid as moditier, 2 ml. ofa solution of mg. glucose oxidase and 75 mg. peroxidase in 100 ml. of a 0.1M phosphate buffer of pH 5.6 and l ml. glucose solution are mixed with 1 ml. of the indicator slurry and stirred up. There were obtained the color reactions set out in the following table.
• Diagnostic agent for use in the analytical determination of (a) hydroperoxide and substances which react with the liberation of hydroperoxide or (b) peroxidase and peroxidatively active substances which agent comprises (a) a member selected from the group consisting of peroxidase and peroxidatively active substances; or (b) a member selected from the group consisting of hydroperoxide and substances which react with the liberation of hydrogen peroxide; respectively, and a chromogen of the formula wherein R is lower alkyl; R is hydrogen or alkyl and, together with Y, can represent a fused benzene or napthalene nucleus; X is sulfur, oxygen, an alkylated imino group, or a vinylene radical; and Y is a methine radical which, together with R,, can also form a benzene or naphthalene nucleus.
• Diagnostic agent as claimed in claim 1 for use in the determination of hydroperoxide or a substance which reacts with the liberation of hydrogen peroxide which agent comprises (l) a member selected from the group consisting of peroxidase and a peroxidatively active substance, and (b) a chromogen as defined in claim 1.
• Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V' is hydrogen or amino or hydroxyl; W is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as'with the alkali metal salts thereof.
• Diagnostic agent as claimed in claim 1 for use in the determination of peroxidase or peroxidatively active substances which agent comprises (1) a member selectedfrom the group consisting of hydrogen peroxide and substances forming hydrogen peroxide and (2) a chromogen as defined in claim 1.
• Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V is hydrogen or amino or hydroxyl; W is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as with the alkali metal salts thereof.
• Diagnostic agent as claimed in claim 1 in the form of a test paper impregnated with said diagnostic agent.
• Diagnostic agent as claimed in claim 1 in the form of a test film impregnated with said diagnostic agent.
• Ar is a benzene or naphthalene nucleus; V is amino or hydroxyl; V is hydrogen or amino or hydroxyl; W is amino or a carboxylic acid or sulfonic acid group; and W is hydrogen or amino, or a carboxylic acid or sulfonic acid group; as well as with the alkali metal salts thereof.
• Process for the analytical determination of (a) hydroperoxide and a substance which reacts with the liberation of hydroperoxide or (b) peroxidase and a peroxidatively active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 9, and producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid.
• chromogen (l) is l-[ l-methylbenzo-(f)quinoline-2-one1-2- [3-ethyl-benzoxazol-2-one l-azine.
• Process for the analytical determination of (a) hydroperoxide and a substance which reacts with the liberation of hydroperoxide or (b) peroxidase and a peroxidatively active substance which comprises contacting a liquid containing the substance of interest with a reagent comprising a diagnostic agent according to claim 1 and producing a color with respect to said liquid as visual evidence of the presence of said substance of interest in said liquid.
• chromogen is a compound having the formula:
• R is lower alkyl
• R is hydrogen or alkyl, preferably lower alkyl, or, together with Y, represents a T fused benzene or naphthalene nucleus
• X is sulfur or oxygen i E or an alkylated imino group, preferably carrying lower I l alkyl groups, or a vinylene radical
• Y is a :nethina radical which, together with R2, can also form a benzene or naphthalene nucleus

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
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• Genetics & Genomics (AREA)
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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

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Citations (1)

• 1970
  • 1970-03-20 US US21493A patent/US3627697A/en not_active Expired - Lifetime
  • 1970-04-06 CA CA079362A patent/CA919562A/en not_active Expired
  • 1970-04-07 FI FI700969A patent/FI50912C/fi active
  • 1970-04-07 CH CH513570A patent/CH530638A/de not_active IP Right Cessation
  • 1970-04-07 GB GB1252725D patent/GB1252725A/en not_active Expired
  • 1970-04-08 FR FR7012660A patent/FR2043073A5/fr not_active Expired
  • 1970-04-08 AT AT322970A patent/AT294015B/de not_active IP Right Cessation
  • 1970-04-08 SE SE04769/70A patent/SE361361B/xx unknown
  • 1970-04-08 NL NL7005007A patent/NL7005007A/xx not_active Application Discontinuation
  • 1970-04-08 ZA ZA702355A patent/ZA702355B/xx unknown
  • 1970-04-09 JP JP3045670A patent/JPS5344834B1/ja active Pending

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